Cytokine induction of selectin ligands on cells

ABSTRACT

Methods and compositions for treating cells with cytokines are provided herein.

RELATED APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No.11/779,650, filed Jul. 18, 2007, which claims priority to U.S.Provisional Application No. 60/831,525, filed Jul. 18, 2006, which isincorporated herein by reference in its entirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

The work described herein was funded, in part through a grant from theNational Institutes of Health (grant RO1 HL060528). The United Statesgovernment has certain rights in the invention.

FIELD OF THE INVENTION

This invention relates to methods and compositions for modulating cellexpression of glycoproteins and glycolipids. The invention alsodescribes reagents and methods of modulating E-selectin ligand activity,e.g., in cases where cytokine-induced E-selectin ligand expression areassociated with adverse effects.

BACKGROUND

The capacity to direct migration of blood-borne cells to a predeterminedlocation (“homing”) has profound implications for a variety ofphysiologic and pathologic processes. Recruitment of circulating cellsto a specific anatomic site is initiated by discrete adhesiveinteractions between cells in flow and vascular endothelium at thetarget tissue(s).

Selectin-mediated interactions are critical not only for the rapid andefficient recruitment of leukocytes at a site of injury, but for steadystate, tissue-specific homing as illustrated in: (1) lymphocyte homingto peripheral lymph nodes, (2) cutaneous tropism of human skin-homingT-cells and (3) hematopoietic progenitor cell (HPC) entry into bonemarrow.

SUMMARY

The invention is based, in part, on the discovery of methods andcompositions for modulating the expression or activity of glycosylationenzymes in a cell. The methods increase the expression or activity of aglycosylated cell-surface molecule, such as a glycolipid or glycoprotein(e.g., a glycolipid or glycoprotein that binds a selectin). Theglycosylation enzyme is a glycosyltransferase such asα2,3-sialyltransferase (ST3GalIV), leukocyte α1,3-fucosyltransferases(FucT-IV, FucT-VII or FucT-IX), or glycosyltransferase core 2β1-6N-acetylglucosaminyl transferase (C2GnT1 or C2GlcNAcT1) or a glycosidasesuch sialidase. The methods and compositions described herein areparticularly useful for augmenting selectin ligand or lewis antigen(e.g. CDS15) expression or activity on various cell types, and can beapplied to enhance the engraftment and/or tissue-regenerative potentialof cells. Accordingly, in one aspect, the invention features a methodfor treating a cell by contacting the cell with one or more cytokinesthat increase the expression or activity of a glycosyltransferasepolypeptide or glycosidase polypeptide in a cell. For example the cellis contacted with two, three, four, five or more cytokines. The methodincreases cell-surface expression or activity of a selectin ligand, alewis antigen (e.g., lewis x), a VIM-2 epitope or a HECA-452-reactiveepitope on the cell. A selectin ligand is a glycoprotein or aglycolipid. For example the selectin ligand is an E-selectin ligand, anL-selectin ligand, a P-selectin ligand. In various embodiments, thecytokine increases the cell-surface expression or activity of anE-selectin ligand on the cell. The E-selectin ligand is, for example,Hematopoietic Cell E-/L-selectin Ligand (HCELL), or the ˜65 kDaE-selectin described herein. In various embodiments, the methodincreases the affinity of the cell for a selectin.

The cell can be a hematopoietic cell, such as a hematopoietic stem cell,e.g., a CD34+ hematopoietic stem cell, a peripheral blood leukocyte, alymphocyte, or a myeloid cell, such as an immature myeloid cell. Othertypes of cells, including non-hematopoietic cells, may also be treatedaccording to the methods. Appropriate non-hematopoietic cells express areceptor for the cytokine of interest. For example, glial and neuronalcells express receptors for granulocyte colony stimulating factor(G-CSF). Neurons are also sensitive to macrophage colony-stimulatingfactor (M-CSF) and interleukin-3 (IL-3).

In various embodiments, the cytokine modulates selectin ligandexpression selectively, on a particular type of cell (e.g., the cytokineselectively acts on hematopoietic cells or another subset of cells). Invarious embodiments, the cell contacted with the cytokine is not a Tcell.

In various embodiments, a plurality of cells is provided. A cell can beprovided ex vivo, or in vivo. The cell can also be contacted with thecytokine in vitro.

The cytokine is contacted with the cell in a concentration range of1-1,000 ng/ml, 1-100 ng/ml, 1-50 ng/ml, 1-25 ng/ml, or 1-10 ng/ml.

Suitable cytokines include those that modulate the expression oractivity of a selectin ligand and/or enzymes that modulate theselectin-binding activity of a selectin ligand, such as carbohydratemodifying enzymes such that expression of selectin-binding epitopesincreases. In one embodiment, the cytokine is granulocyte colonystimulating factor (G-CSF). Alternatively, the cytokine is granulocytemacrophage colony stimulating factor (GM-CSF), macrophage colonystimulating factor (M-CSF), interleukin-3 (IL-3)/multi colonystimulating factor (Multi-CSF), transforming growth factor β (TGFβ), aninterferon, a chemokine, an interleukin or a tumor necrosis factor. Aninterleukin includes, for example, IL-1, IL-2, IL-4, IL-5, IL-6, IL-7,IL-8, IL-9, IL-10, IL-11, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18,IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28,and IL-29.

The cell is treated ex vivo, and the method further includesadministering the cell, or a plurality of cells, to a subject, e.g., asubject in need of treatment for tissue injury, and/or cell as part of ahematopoietic stem cell transplantation protocol. Optionally, the cellswhich are administered are a selected subset of cells (e.g., a subsetwhich have been selected based on a particular phenotype or expressionof a cell surface marker, such as a stem cell marker). The cells may beenriched for those which express high levels of the selectin ligand.Additionally, the method includes selecting a subpopulation of cells(e.g., a subpopulation of leukocytes, or stem cells, such ashematopoietic stem cells, or stem cells which support regeneration of adesired tissue) prior to contacting the cells with a cytokine.

In another aspect, the invention features a kit for treatment of a cellto increase its engraftment and/or regenerative potential. The kitincludes, for example: a composition comprising a cytokine (e.g.,G-CSF), and instructions for use of the cytokine to treat a cell underconditions in which the cytokine increases the cell-surface expressionor activity of a selectin ligand polypeptide on the cell, therebyincreasing the engraftment and/or regenerative potential of the cell.

In another aspect, the invention features a composition comprising aglycoprotein isolated from granulocyte colony stimulating factor-treatedperipheral blood leukocytes, wherein the glycoprotein is approximately65 kDa, is reactive with monoclonal antibody HECA-452, and is a ligandfor E-selectin. The glycoprotein may be purified or isolated. In variousembodiments, the glycoprotein includes other features described herein.The invention also includes derivatives of the 65 kDa glycoprotein whichcompete with the natural 65 kDa glycoprotein for binding to E-selectin.

In another aspect, the invention features a method for treating asubject who has received, or is scheduled to receive granulocyte colonystimulating factor (G-CSF). The method include: administering to thesubject an agent which inhibits a selectin-mediated (e.g.,E-selectin-mediated) interaction with a selectin ligand. In variousembodiments, the method reduces side effects due to administration ofG-CSF, such as enhanced leukocyte-endothelial interactions that areassociated with adverse inflammatory reactions. The agent is, forexample, an antibody or antigen-binding fragment thereof, a smallinterfering RNA, or an antisense oligonucleotide. In variousembodiments, the agent inhibits the expression or activity of aglycosyltransferase and/or interferes with carbohydrate synthesis. Theagent may also be a soluble carbohydrate or glycosylated polypeptidewhich directly inhibits an interaction between a selectin and a selectinligand, e.g., by competition for binding to either the selectin or theselectin ligand. Examples of suitable agents include soluble mimetics ofselectin ligands. The agent can be a soluble form of a natural selectin,selectin ligand or a derivative thereof which exhibits enhanced affinityfor the selectin ligand or for the selectin, respectively, or to both.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In the case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and are notintended to be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 A-G illustrates ML possess enhanced binding to E-selectinrelative to native leukocytes (NL). Human umbilical vein endothelialcells (HUVEC) were stimulated with TNF-αfor 4-6 hrs. Subsequently, ML orNL were perfused in a parallel plate apparatus over HUVEC at 1.5dyne/cm². In certain instances, HUVEC were pretreated with a mAb toE-selectin prior to use in adhesion assays. (A) Primary tethering and(B) average rolling velocity of ML or NL on HUVEC was determined. “Stim”indicates stimulation (+) or no stimulation (−) of HUVEC with TNF-α for4-6 hours prior to the assay; α-E-sel indicates pretreatment (+) or nopretreatment (−) of HUVEC with a function blocking mAb to E-selectin,(68-5411); EDTA indicates presence (+) or absence (−) of 5 mM EDTA inthe assay buffer. Values are means±SE of n≧4 different runs. * indicatesstatistically significant difference (p<0.05). C-D Are bar graph resultsof ML or NL perfused over CHO-E at 1 dyne/cm². In certain instances,CHO-E were pretreated with a function blocking mAb to E-selectin priorto use in adhesion assays. (C) Primary tethering and (D) average rollingvelocity of ML or NL on CHO-E was determined. α-E-sel indicatespretreatment (+) or no pretreatment (−) of CHO-E with a functionblocking mAb to E-selectin, (68-5411); EDTA indicates presence (+) orabsence (−) of 5 mM EDTA in the assay buffer; Values are means±SE of n≧5different runs. * indicates statistically significant difference(p<0.05). E-G Show results of ML and NL that were treated with DiD exvivo and injected into the tail vein of mice 5-6 hours after stimulationof one ear locally with TNF-α. (E) Represents the average rollingvelocity of ML or NL on inflamed vascular endothelium in TNF-α-treatedears of mice. Values represent means±SE of 10-20 leukocytes per mousewith n=3 for each group. (F) Shows representative images of the adhesiveinteractions of NL (top) and ML (bottom) with inflamed vascularendothelium in TNF-α-treated ears of two separate mice. Minimal adhesiveinteractions were observed in control PBS-treated ear (not shown). (G)is a bar graph showing adherent leukocytes per field of view. Valuesrepresent means±SE of 15-25 fields of view per mouse with n=3 for eachgroup. * indicates statistically significant difference (p<0.05).

FIGS. 2A-E illustrates how ML express multiple HECA-452-reactiveE-selectin glycoprotein ligands. (A) Shows HECA-452 blots of cell lysatefrom (unfractionated) buffy coat of ML or NL resolved on a reducing4-20% SDS-PAGE gel. (B) Shows HECA-452 blots of membrane preparations ofmononuclear fraction of G-CSF mobilized peripheral blood (MPB)leukocytes (20 μg; ML-M), G-CSF mobilized granulocytes (40 μg; ML-G);native peripheral blood mononuclear cells (40 μg; PBMC) or nativeperipheral blood polymorphonuclear leukocytes (20 μg; PMN) resolved on areducing 4-20% SDS-PAGE gel. Note the distinct presence ofHECA-452-reactive ˜100 kDa and ˜65 kDa bands in G-CSF mobilizedleukocytes. In all experiments, rat IgM isotype control blots performedin parallel lacked staining. Results presented are representative ofobservations on numerous HECA-452 blots from numerous clinical samplesof G-CSF MPB. (C) Is a bar graph of CHO-E that were perfused overSDS-PAGE immunoblots of HECA-452 reactive membrane glycoproteins of ML-Mat 0.6 dyne/cm² and the number of interacting cells/mm² was tabulated asa function of molecular weight. The background binding was subtractedand the results compiled into an adhesion histogram. Results presentedare representative of multiple runs and multiple observations onnumerous HECA-452 blots of membrane preparations of ML-M. (D) Membranepreparations of ML-M (20 μg) were resolved on a reducing 4-20% SDS-PAGEgel and immunoblotted with E-selectin-Ig (E-Ig) chimera in the presenceof Ca²⁺. The E-Ig chimera reactive glycoproteins at ˜220 kDa, ˜130 kDa,˜100 kDa, and ˜65 kDa corresponded exactly with the proteins stained byHECA-452. Result shown is representative of multiple observations onnumerous E-Ig blots of various clinical specimens of ML-M. (E) Shows theresults of E-Ig used to immunoprecipitate E-selectin ligands frommembrane preparations of ML-M, and the resolved immunoprecipitate wasblotted with HECA-452. HECA-452 stained E-Ig immunoprecipitated materialat ˜220 kDa, ˜130 kDa, ˜100 kDa and ˜65 kDa bands.

FIGS. 3A-F illustrates the characterization of PSGL-1, HCELL and a novelE-selectin ligand on ML. (A) Shows the membrane preparations of ML-Mwere resolved on a reducing 4-20% SDS PAGE gel, and immunoblotted withKPL-1, an antibody to PSGL-1. The bands at ˜220 kDa and ˜130 kDacorresponded with the HECA-452-reactive membrane glycoproteins on ML-M.Mouse IgG_(1,k) isotype control blots performed in parallel lackedstaining. (B) Shows that KPL-1 was used to immunoprecipitate PSGL-1, andthe resolved immunoprecipitate was blotted with either HECA-452 orKPL-1. (C) Illustrates the membrane preparations of ML-M were resolvedon a reducing 4-20% SDS PAGE gel, and immunoblotted with Hermes-1, anantibody to CD44. The band at ˜100 kDa corresponded with theHECA-452-reactive membrane glycoprotein on ML-M. Rat IgG isotype controlblots performed in parallel lacked staining. (D) Hermes-1 was used toimmunoprecipitate CD44, and the resolved immunoprecipitate was blottedwith either HECA-452 or another anti-human CD44 mAb, 2C5. (E) Is a graphshowing the results of mAb Dreg-56 used to determine L-selectinexpression on ML and NL using flow cytometry. mIgG_(1,k) served asisotype control for Dreg-56. Results shown are typical of multipleclinical specimens. (F) Shows the results of L-selectinimmunoprecipitated from biotinylated ML-M (MPB) and KG1a cells wasresolved on a reducing SDS-PAGE gel, and immunoblotted with horseradishperoxidase (HRP) conjugated strepavidin. Note that the ˜80 kDaL-selectin band is present in KG1a cells and absent in ML-M.

FIGS. 4A-B illustrates ML possess enhanced levels ofglycosyltransferases ST3GalIV, FucT-IV and FucT-VII. Total RNA fromequal numbers of ML and NL was subjected to RT-PCR followed by PCRamplification of pairs of cDNAs for ST3GalIV, FucT-IV, FucT-VII and thehousekeeping gene GAPDH. (A) Shows the net intensity of amplified bandswas normalized to the net intensity of respective GAPDH controls. Allvalues are means±SE of at least 3 different experiments. (B) Shows thattypical blots of PCR amplified products from NL and ML RNA arepresented.

FIGS. 5A-C shows HCELL and ˜65 kDa glycoprotein are major E-selectinligands on ML. (A) Are graphs of PSGL-1, CD44 and HECA-452 antigen(s)expression determined on untreated (native) and OSGE-treated ML usingflow cytometry. mAbs KPL-1 and F10-44-2 were used to determineexpression of PSGL-1 and CD44, respectively. mIgG_(1,k) and mIgM servedas isotype controls for KPL-1 and F10-44-2, respectively. rIgM served asisotype control for HECA-452. Note that OSGE treatment abrogates surfaceexpression of PSGL-1 and has minimal effect on expression of CD44 orHECA-452 antigen(s). Results shown are representative of 2 differentexperiments. (B) E-Ig blots of cell lysates from equal numbers ofuntreated (−) or 30 μg/ml OSGE treated (+) ML resolved on a reducing4-20% SDS-PAGE gel. Note that OSGE treatment markedly abrogatesE-selectin binding capacity of PSGL-1 with little to no effect onE-selectin binding capacity of HCELL and ˜65 kDa E-selectin ligand. (C)Is a bar graph showing the results of HUVEC that were stimulated withTNF-α for 4-6 hrs. Subsequently, untreated (−) or OSGE (+) treated MLwere perfused over HUVEC at 1.5 dyne/cm². Primary tethering of untreatedor OSGE-treated ML on HUVEC was determined. Values are means±SE of n≧3different runs.

FIGS. 6A-E shows in vitro G-CSF treatment of human bone marrow (BM)cells up-regulates the expression of HCELL and HECA-452-reactive ˜65 kDaglycoprotein. (A) (left) HECA-452 and (right) E-Ig blots of cell lysatesfrom human BM mononuclear cells (BM-MNC) or BM CD34+/lineage− cells(Lin−) resolved on a reducing 4-20% SDS-PAGE gel. (B) (left) HECA-452and (right) E-Ig blots of cell lysates from Band 1 (B1), Band 2 (B2) andBand 3 (B3) ML resolved on a reducing 4-20% SDS-PAGE gel. Note thepresence of HCELL and HECA-452 reactive ˜65 kDa glycoproteinpredominantly in Band 1 and Band 2 cells. (C) (left) HECA-452 and(right) E-Ig blots of cell lysates from untreated (−) or 72 hr. G-CSFtreated (+) Band 1, Band 2 and Band 3 human BM cells resolved on areducing 4-20% SDS-PAGE gel. Note that G-CSF treatment results in amarked up-regulation of ˜100 kDa HCELL and HECA-452-reactive ˜65 kDaglycoprotein predominantly in immature myeloid cells. (D-E) Total RNAfrom equal numbers of untreated and G-CSF-treated Band 2 BM cells wassubjected to RT-PCR followed by PCR amplification of pairs of cDNAs forST3GalIV, FucT-IV, FucT-VII and the housekeeping gene GAPDH. (D) The netintensity of amplified bands was normalized to the net intensity ofrespective GAPDH controls. All values are means±SE of at least 3different experiments. (E) Shows typical blots of PCR amplified productsfrom untreated (U) and G-CSF-treated (G) Band 2 BM cells RNA arepresented.

FIG. 7 illustrates that there is no distinct difference in the surfaceexpression of integrin-type homing receptors (e.g., LFA-1 (CD11a/CD18;αLβ2) and VLA-4 (CD49d/CD29; α4β1)) and chemokine receptor CXCR4 on MLand NL. CD11a, CD18, CD29, CD49d and CXCR4 expression was determined onML and NL using flow cytometry. mAbs 25.3, 7E4, HUTS-21, HP2/1 and 12G5were used to determine expression of CD11a, CD18, CD29, CD49d and CXCR4,respectively. mIgG_(1,k) served as an isotype control for 25.3, 7E4 andHP2/1 and mIgG_(2a) served as an isotype control for HUTS-21 and 12G5.Results shown are representative of 2 separate experiments.

FIG. 8 shows HECA-452-reactive glycoproteins of ML are sensitive tosialidase treatment. Membrane preparations of ML-M (10 μg) were treatedwith sialidase (+) or buffer treated (−), resolved on a reducing 4-20%SDS-PAGE gel and immunoblotted with HECA-452. Absence of stainingfollowing sialidase digestion confirms specificity of HECA-452 stainingfor sialofucosylated carbohydrate modifications.

FIGS. 9A-B illustrate E-Ig-reactive glycoproteins of ML do not stain inthe presence of EDTA or with control human-Ig. Membrane preparations ofML-M (20 μg) were resolved on a reducing 4-20% SDS-PAGE gel andimmunoblotted with (A) E-selectin-Ig (E-Ig) chimera in the presence of10 mM EDTA or (B) human-Ig. The E-Ig chimera reactive glycoproteins at˜220 kDa, ˜130 kDa, ˜100 kDa, and ˜65 kDa (FIG. 2 d) do not stain in thepresence of EDTA or with control human-Ig. Results shown are typical of2 separate experiments.

FIG. 10 shows HECA-452-reactive ˜65 kDa E-selectin ligand does notappear to be related to PSGL-1 or CD44. PL2 and 2C5 were used toimmunoprecipitate PSGL-1 and CD44, respectively, from membranepreparations of ML-M. Mouse IgG₁ was used as an isotype control forimmunoprecipitation. The immunoprecipitated materials were resolved on areducing 4-20% SDS PAGE gel, and immunoblotted with HECA-452. Note thatPL-2 and 2C5 did not immunoprecipitate the HECA-452-reactive ˜65 kDaglycoprotein.

FIGS. 11A-B illustrates ML possess diminished binding to P-selectinrelative to NL. ML or NL were perfused over CHO-P at 1.0 dyne/cm². (A)Illustrates primary tethering and (B) shows rolling velocity of ML or NLon CHO-P was determined. Values are means±SE of n≧6 different runs. *indicates statistically significant difference (p<0.05).

FIGS. 12A-B illustrates G-CSF treatment enhances the capability of humanBM cells to adhere to endothelial E-selectin under physiologic flowconditions. HUVEC were stimulated with IL-1β for 4-6 hrs. Subsequently,untreated or 72 hr. G-CSF-treated Band 2 cells were perfused over HUVECat 1.5 dyne/cm². In certain instances, stimulated HUVEC were pretreatedwith a mAb to E-selectin prior to use in adhesion assays. (A)Illustrates primary tethering and (B) shows rolling velocity ofuntreated or G-CSF-treated cells on stimulated HUVEC. Stim. indicatespretreatment (+) or no pretreatment (−) of HUVEC with IL-1β for 4-6hours prior to the assay; α-E-sel indicates pretreatment (+) or nopretreatment (−) of HUVEC with a function blocking mAb to E-selectin,(68-5411); EDTA indicates presence (+) or absence (−) of 5 mM EDTA inthe assay buffer. Values are means±SE of n≧5 different runs. * indicatesstatistically significant difference (p<0.05).

FIG. 13 illustrates that G-CSF treatment increases Fuct-IX expression innormal progenitors and in mobilized peripheral blood. Lane 1 are normalcells isolated from bone marrow from healthy donor. Lane 2 are G-CSFtreated normal progenitors isolated from bone marrow from healthy donor.Lane 3 are untreated cells isolated from G-CSF mobilized blood. Lane 4are G-CSF treated cells isolated from G-CSF mobilized blood.

FIG. 14 is a bar graph showing that G-CSF treatment of human myeloidbone marrow cells increases sialidase activity. Sialidase activity fromhuman myeloid cells (obtained from bone marrow of normal subjects),before and after treatment with 10 ng/mL of G-CSF for 24 hours wasmeasured using 4-MU-NANA as substrate, * indicates significantlydifferent (p≦0.05).

FIG. 15 is a bar graph illustrating that inhibition of sialidase withDANA blunts G-CSF-induced increase in CD15. Bone marrow cells weretreated with 10 ng/mL of G-CSF for 5 days in the presence or absence of100 mM DANA and expression of CD15 was analyzed by flow cytometry.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION

The invention is based in part on the surprising discovery thatcytokines can induce the expression of glycosylation enzymes in a cell.Specifically, G-CSF induced the expression of glycosyltranferases in acell which resulted in the increased expression of hematopoietic cellE-/L-selectin ligand (HCELL) and an approximately 65 kDa E-selectinligand. Additionally, G-CSF induced the expression of sialidase whichresulted in an increased expression of CD15.

Granulocyte colony stimulating factor (G-CSF) is widely used clinicallyto augment neutrophil recovery after myelosuppressive chemo/radiotherapyand for mobilizing bone marrow (BM) hematopoietic progenitors for use inhematopoietic stem cell transplantation (HSCT)¹. Though generallyconsidered safe, there are increasing observations that G-CSFadministration can promote leukocyte-endothelial adhesive interactionsresulting in vascular and inflammatory complications²⁻¹³. Indeed, G-CSFadministration has been shown to (i) recruit neutrophils to lungvasculature resulting in respiratory distress syndrome⁴⁻⁶, (ii)stimulate granulocyte adherence to endothelium resulting in anginapectoris/myocardial infarct⁷, (iii) cause neutrophil infiltration indermal vessels^(8,9) leading to development of cutaneousleukocytoclastic vasculitis¹⁰, (iv) intensify arthritic symptoms¹¹ and(v) precipitate sickle cell vaso-occlusion¹². Though G-CSFadministration may have favorable effects on myocardial recoveryfollowing infarct in preclinical models¹⁴, a recent report of G-CSFadministration in patients with coronary artery disease revealed astriking incidence of cardiac ischemic complications¹³. A betterunderstanding of the molecular basis of the enhancedleukocyte-endothelial interactions accompanying clinical G-CSFadministration could yield strategies to prevent these complications.

Despite decades of clinical observations on G-CSF biology, the effectsof G-CSF administration on leukocyte membrane molecules that bindendothelial counter-receptors under hemodynamic shear conditions areunknown. In this study, the capacity of leukocytes mobilized by G-CSF(ML) to bind to inflamed (TNF-α-stimulated) endothelium was evaluated.Parallel plate assays conducted under physiologic flow conditions andintravital microscopy of a murine inflammation model each showed that,compared to NL, ML display markedly increased adhesive interactions withinflamed endothelium, mediated by enhanced E-selectin receptor/ligandinteractions. ML expressed the potent E-selectin ligand HCELL andanother heretofore unrecognized E-selectin glycoprotein ligand of ˜65kDa, and possessed enhanced levels of critical glycosyltransferases(ST3GalIV, FucT-IV, FucT-VII and FucT-IX) rendering E-selectin ligandactivity. Enzymatic removal of PSGL-1 revealed that these novel ligandsare the principal mediators of the robust ML adhesion to vascularE-selectin. Treatment of normal human BM cells with clinically-relevantserum levels of G-CSF in vitro increased the expression of pertinentglycosyltransferases directly inducing the expression of these twoligands and resulting in enhanced E-selectin-mediated endothelialbinding. Collectively, these results provide first evidence thatenhanced leukocyte-endothelial interactions following G-CSFadministration is mediated by G-CSF-induced expression ofcounter-receptors for vascular E-selectin among circulating myeloidcells and offer mechanistic insights on the molecular basis ofG-CSF-induced increased E-selectin ligand activity.

In this study, no distinct change in HECA-452 reactivity of PSGL-1 on MLcompared to that of NL (FIG. 2 a) was observed. Furthermore, OSGEtreatment of ML decreased PSGL-1 expression and function withoutaffecting the overall E-selectin binding capacity of ML (FIG. 4).Interestingly, ML possessed diminished P-selectin binding compared to NL(FIG. 11) suggesting that PSGL-1 function is also altered on ML.Consistent with prior studies³⁴, we observed marked decrease in surfaceL-selectin expression on circulating leukocytes following G-CSFadministration (FIG. 3 e), excluding a role for L-selectin as anE-selectin ligand on ML.

In contrast to the broad distribution of PSGL-1, HCELL ischaracteristically found primarily on normal human BM CD34+progenitors^(28,29). Herein, biochemical studies of ML show that G-CSFadministration results in robust HCELL expression on circulating(mobilized) myeloid cells, most prominently on immature myeloid cells(FIG. 6 b). The observed augmented HCELL expression is a direct effectof G-CSF, as exposure to pharmacologically-relevant G-CSF concentrationsin vitro^(44,45) results in increases in glycosyltransferases and theelaboration of HECA-452-reactive glycosylations rendering the HCELLphenotype on immature human BM myeloid cells (FIG. 6 c). Though lowlevel expression of HCELL on native circulating human PMNs is observedoccasionally, G-CSF had only a variable effect on inducing HCELLexpression among mature myeloid BM cells. Importantly, HCELL is notexpressed on lymphocytes in BM or in blood, and it is not induced onlymphocytes by G-CSF treatment.

In addition to induction of HCELL, G-CSF administration in vivo and invitro also induces the expression of a HECA-452-reactive ˜65 kDaglycoprotein. The results of blot-rolling assays, Western blot stainingwith E-Ig, and immunoprecipitation with E-Ig of membrane preparations ofML show that the ˜65 kDa glycoprotein is a high affinity E-selectinligand. The G-CSF-induced ˜65 kDa E-selectin ligand does not appear tobe a glycoform of other previously described E-selectin ligands (PSGL-1,CD44, and L-selectin (FIG. 3)), and thus represents a novel E-selectinligand. Importantly, this ˜65 kDa glycoprotein is found predominantlyamong cells within the mononuclear fraction of ML (ML-M cells) (FIG. 6b). Although the various leukocyte subset(s) that express this ˜65 kDaglycoprotein are currently unknown and warrant further investigation,its expression is conspicuously absent from lymphocytes (both T- andB-cells) within the ML-M fraction.

From a clinical perspective, defining the molecular mechanism(s) ofG-CSF-induced vascular and inflammatory complications is of paramountimportance as healthy individuals are increasingly being exposed to thisagent to serve as donors for hematopoietic stem cell therapy (HSCT). Itis plausible that G-CSF may preferentially mobilize subset(s) of myeloidcells which express high affinity E-selectin ligands. Consistent withthis notion are clinical observations that G-CSF-associated adverseevents occur in parallel to increases in leukocyte numbers^(4,9,12).However, not all donors with high leukocyte counts will exhibitcomplications, suggesting that some individuals may be particularlysusceptible to G-CSF-induced vascular and inflammatory problems. To someextent, this may reflect variabilities in the capacity of G-CSF toinduce E-selectin ligand expression on circulating myeloid cells and/orresponsiveness of the endothelial cells of G-CSF recipients to theinduction of E-selectin expression. However, in multiple clinical MLcollections, it was observed that G-CSF uniformly increased E-selectinligand activity of circulating leukocytes and upregulated the expressionof the E-selectin ligands HCELL and the HECA-452-reactive ˜65 kDaglycoprotein. The cumulative effect of these additional, G-CSF-inducedE-selectin ligands to that of natively expressed PSGL-1 on immature (andmature) myeloid cells may prime these circulating cells to adhere toinflamed/ischemic endothelium, consistent with emerging clinicalexperiences/reports raising warnings for the use of G-CSF in individualswith known or suspected inflammatory or cardiovascular diseases.

Under physiologic blood flow conditions, leukocytes initially makecontact on the vessel surface by engagement of counter-receptors forrelevant endothelial molecules that mediate shear-resistantinteractions^(15,16). One of the principal effectors of theseinteractions is E-selectin, which is an inducible endothelial moleculeexpressed at sites of inflammation^(16,17) that binds sialofucosylatedcarbohydrate ligands expressed on leukocytes¹⁸. An expanding body ofevidence causally links upregulated E-selectin expression to vascularcomplications of G-CSF administration¹⁹⁻²². Notably, the receptor forG-CSF is expressed on endothelium²³ and G-CSF directly inducesE-selectin expression on endothelial cells in culture²⁴. However, thereis little information on whether G-CSF administration modifiesE-selectin ligand expression on mobilized, circulating leukocytes.

These findings provide new perspectives on selectin ligands and on thebiology of G-CSF, and indicate that induction of potentcounter-receptors for E-selectin is contributory to the vascular andinflammatory complications observed with the use of this agent.

Methods of Increasing Glycosylation Enzyme Expression or Activity

Glycosylation enzyme expression or activity in a cell is increased bycontacting a cell with a cytokine. Glycosylation enzymes include forexample glycosytransferases or glycosidases. Glycosyltransferasecatalyze the transfer of glycosyl groups to an acceptor and areresponsible for the formation of glycosidic bonds. In contrast,glycosidases catalyze hydrolysis of the glycosidic linkage and areresponsible for the trimming of glycans during carbohydrate synthesis.Glycosyltransferases and glycosidases form the major catalytic machineryfor the synthesis and breakage of glycosidic bonds involved incarbohydrate synthesis.

Glycosyltransferase includes for example, core 1 glycosyltransferase(e.g., β-3-galactosyltransferase); core 2 glycosyltransferase (e.g.,N-acetylglucosaminyltransferases such as β-(1,6)N-acetylglucosaminyltransferase, GnTI, GnTII, GnTIII, GlcNAcT1;sialyltransferases (e.g., α-sialyltransferases, such as α-2,3sialyltransferases (ST3GalIV) α-2,6 sialyltransferases, andβ-sialyltransferases); fucosyltransferases (e.g., α-fucosyltransferases,such as α-1,3 fucosyltransferases (FucT IV, FucT VII, FucT IX) andβ-fucosyltransferases; galactosyltransferases; (e.g.,α-galactosyltransferases, such as α1,3 galactosyltransferases andβ-galactosyltransferases); mannosyltransferases (e.g.,α-mannosyltransferases and β-mannosyltransferases), orN-acetylgalactosaminyltransferases (e.g., α-(1,3)N-acetylgalactosaminyltransferase and β-(1,4)N-acetylgalactosaminyltransferases).

Glycosidases include for example glucosidases, mannosidases,fucosidases, sialidases, galactosidases, xylanases, lactases, amylases,chitinases, sucrases, maltases, neuraminidases, invertases,hyaluronidase or lysozymes.

Cytokines include for example, granulocyte colony stimulating factor(G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF),macrophage colony stimulating factor (M-CSF), interleukin-3 (IL-3)/multicolony stimulating factor (Multi-CSF), transforming growth factor β(TGFβ), an interferon, a chemokine, a tumor necrosis factor or aninterleukin, such as interleukin-1 (IL-1) IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-14, IL-16,IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26,IL-27, IL-28, and IL-29.

Cells to be treated include any cell capable of expressing aglycoprotein or a glycolipid. The cell is a hematopoietic cell, such asa hematopoietic stem cell, e.g., a CD34+ hematopoietic stem cell, a bonemarrow cell, a neutrophil, a peripheral blood leukocyte, a lymphocyte,or a myeloid cell, such as an immature myeloid cell. Alternatively, thecell is a non-hematopoietic cells such as a liver cell, a lung cell, apancreatic cell, a cardiac cell, a gastric cell or a kidney cell. Thecell is contacted in vitro, ex vivo or in vivo.

Alternatively, the cell is contacted with a cytokine in an amountsufficient to increase expression or activity of a glycoprotein orglycolipid. The glycoprotein or glycolipid is cytosolic or cell membraneprotein or lipid. By increased expression or activity of a glycoproteinor a glycolipid it is meant that the cell expresses a greater amount ofthe glycoprotein or a glycolipid as compared to a cell that has not beencontacted with the cytokine or that the affinity of the cell for theligand of the glycoprotein or a glycolipid is increased. For example,the treated cell has an increase affinity for a selectin. Affinity ismeasured by methods known in the art.

For example, cell surface expression of a selectin ligand, or a lewisantigen is increased following treatment of the cell with the cytokine.Selectins include E-selectin, L-selectin or P-selectin. The selectinligands include, for example, MAdCAM1, CD334, PSGL-1, CD24, ESL-1 or theVIM-2 epitope. Preferably, the selectin ligand is, a hematopoietic cellE-/L-selectin ligand (HCELL) or a 65 kDa E-selectin ligand describedherein. Lewis antigens include Le^(a) epitope, the Le^(b) epitope,Le^(x) or the Le^(y) epitope. The Lewis antigen is sialyated.Preferably, cell surface expression of a Le^(x) epitope or the siaylLe^(x) epitope is increased following cytokine treatment of the cell.Exemplary, Le^(x) antigens include CD15 and CD15s.

The invention also provides methods to treat or alleviate the symptomsof a variety of disorders. For example, cells produced by the methods ofthe invention can be administered to a subject to treat a tissue injuryor as part of a stem cell transplant protocol. The cells treatedaccording to the method of the invention have an increasedregenerative/engraftment potential and are useful for a variety oftherapeutic methods including, tissue repair, tissue regeneration, andtissue engineering. By increased regenerative/engraftment potential itis meant that the cell has a greater survival rate after transplantationas compared to an untreated cell.

For example, the cells treated according to the methods of the inventionare useful in bone regeneration, cardiac regeneration, vascularregeneration, neural regeneration and the treatment of ischemicdisorders. Ischemic conditions include, but are not limited to, limbischemia, congestive heart failure, cardiac ischemia, kidney ischemia,ESRD, stroke, and ischemia of the eye. The cells are administered tomammalian subjects, to effect tissue repair or regeneration. The cellsare administered allogeneically or autogeneically.

The cell can be of mesodermal, ectodermal or endoderamal origin.Preferably, the cell is a stem cell. More preferably the cell is ofmesodermal origin. For example, the cell is a hematopoietic progenitorcell.

Included in the invention is a method of increasing the engraftmentpotential of a cell by providing a cell and contacting said cell withone or more cytokines that increases cell-surface expression or activityof a selectin ligand, e.g. HCELL on the cell. The invention furtherprovides a method of increasing levels of engrafted stem cells in asubject, e.g., human, by administering to the subject a cytokine thatincreases cell-surface or expression of a selectin ligand on one or morestem cells in the subject. The cytokine can be administered in vivo, exvivo or in vitro.

The subject is preferably a mammal. The mammal can be, e.g., a human,non-human primate, mouse, rat, dog, cat, horse, or cow. Additionally,the subject suffers from or is at risk of developing a hematopoieticdisorder, e.g, leukemia, cancer, or a tissue injury. A mammal sufferingfrom or at risk of developing a hematopoietic disorder, e.g, leukemia,cancer, or tissue injury can be identified by the detection of a knownrisk factor, e.g., gender, age, prior history of smoking, genetic orfamilial predisposition, attributed to the particular disorder.Alternatively, a mammal suffering from or at risk of developing ahematopoietic disorder, e.g, leukemia, or tissue injury can beidentified by methods known in the art to diagnosis a particulardisorder.

Pharmaceutical Administration and Dosage Forms

The described cells can be administered as a pharmaceutically orphysiologically acceptable preparation or composition containing aphysiologically acceptable carrier, excipient, or diluent, andadministered to the tissues of the recipient organism of interest,including humans and non-human animals. Cell-containing compositions canbe prepared by resuspending the cells in a suitable liquid or solutionsuch as sterile physiological saline or other physiologically acceptableinjectable aqueous liquids. The amounts of the components to be used insuch compositions can be routinely determined by those having skill inthe art.

The cells or compositions thereof can be administered by placement ofthe cell suspensions onto absorbent or adherent material, i.e., acollagen sponge matrix, and insertion of the cell-containing materialinto or onto the site of interest. Alternatively, the cells can beadministered by parenteral routes of injection, including subcutaneous,intravenous, intramuscular, and intrasternal. Other modes ofadministration include, but are not limited to, intranasal, intrathecal,intracutaneous, percutaneous, enteral, and sublingual. In one embodimentof the present invention, administration of the cells can be mediated byendoscopic surgery.

For injectable administration, the composition is in sterile solution orsuspension or can be resuspended in pharmaceutically- andphysiologically-acceptable aqueous or oleaginous vehicles, which maycontain preservatives, stabilizers, and material for rendering thesolution or suspension isotonic with body fluids (i.e. blood) of therecipient. Non-limiting examples of excipients suitable for use includewater, phosphate buffered saline, pH 7.4, 0.15 M aqueous sodium chloridesolution, dextrose, glycerol, dilute ethanol, and the like, and mixturesthereof. Illustrative stabilizers are polyethylene glycol, proteins,saccharides, amino acids, inorganic acids, and organic acids, which maybe used either on their own or as admixtures. The amounts or quantities,as well as the routes of administration used, are determined on anindividual basis, and correspond to the amounts used in similar types ofapplications or indications known to those of skill in the art.

Consistent with the present invention, the cell can be administered tobody tissues, including liver, pancreas, lung, salivary gland, bloodvessel, bone, skin, cartilage, tendon, ligament, brain, hair, kidney,muscle, cardiac muscle, nerve, skeletal muscle, joints, and limb.

The number of cells in a cell suspension and the mode of administrationmay vary depending on the site and condition being treated. Asnon-limiting examples, in accordance with the present invention, about35−300×10⁶ cells are injected to effect tissue repair. Consistent withthe Examples disclosed herein, a skilled practitioner can modulate theamounts and methods of cell-based treatments according to requirements,limitations, and/or optimizations determined for each case.

The preferred suspension solution is Multiple Electrolyte Injection Type1 (USP/EP). Each 100 mL of Multiple Electrolyte Injection Type 1contains 234 mg of Sodium Chloride, USP (NaCl); 128 mg of PotassiumAcetate, USP (C₂H₃KO₂); and 32 mg of Magnesium Acetate Tetrahydrate(Mg(C₂H₃O₂)₂.4H₂O). It contains no antimicrobial agents. The pH isadjusted with hydrochloric acid. The pH is 5.5 (4.0 to 8.0). TheMultiple Electrolyte Injection Type 1 is preferably supplemented with0.5% human serum albumin (USP/EP). Preferably, the cell pharmaceuticalcomposition is stored at 0-12° C., unfrozen.

Indications and Modes of Delivery for Cells

Cells may be manufactured and processed for delivery to patients usingthe described methods where the final formulation is the cells with allculture components substantially removed to the levels deemed safe bythe FDA. It is critical for the cells to have a final viability greaterthan 70%, however the higher the viability of the final cell suspensionthe more potent and efficacious the final cell dose will be, and theless cellular debris (cell membrane, organelles and free nucleic acidfrom dead cells), so processes that enhance cell viability whilemaintaining the substantially low culture and harvest components, whilemaintaining closed aseptic processing systems are highly desirable.

Limb Ischemia

It has been demonstrated that bone marrow-derived cells are used forvascular regeneration in patients with critical limb ischemia,peripheral vascular disease, or Burger's syndrome. The cells deliveredto patients with ischemic limbs, and have been shown to enhance vascularregeneration. Cells are delivered to patients by creating a cellsuspension and removing the cells from the supplied bag or vial in whichthey are delivered. A syringe is used to remove the cell suspension, andthen smaller 0.25 ml to 1 ml individual injection volumes are loadedfrom the main syringe using a syringe adaptor, and then severalindividual injection volumes are delivered via intramuscular injectionto the site of limb ischemia and where vascular formation is required.The cells may be delivered through a wide range of needle sizes, fromlarge 16 gauge needles to very small 30 gauge needles, as well as verylong 28 gauge catheters for minimally invasive procedures.Alternatively, the cells may also be delivered intravascularly andallowed to home to the site of ischemia to drive local tissueregeneration.

Cardiac Regeneration

There are a variety of modes of delivery for driving cardiac tissueregeneration. The cells are delivered intra-vascularly and allowed tohome to the site of regeneration. Alternatively, the cells are also bedelivered directly into the cardiac muscle, either epicardially orendocardially, as well as transvascularly. The cells may be deliveredduring an open-chest procedure, or via minimally invasive proceduressuch as with delivery via a catheter. The cells are delivered to thesepatients by creating a cell suspension and removing the cells from thesupplied bag or vial in which they are delivered. A syringe is used toremove the cell suspension, and then smaller 0.25 ml to 1 ml individualinjection volumes are loaded from the main syringe using a syringeadaptor, and then several individual injection volume are delivered viaintramuscular injection to the site of cardiac ischemia and wherevascular formation is required. The cells may be delivered through awide range of needle sizes, from large 16 gauge needles to very small 30gauge needles, as well as very long 28 gauge catheters for minimallyinvasive procedures.

Spinal Cord Regeneration

There are a variety of ways that cells are used for regeneration afterspinal cord injury (SCI). Cells may be injected directly into the siteof SCI, seeded onto a matrix (chosen from the list below for boneregeneration) and seeded into re-sected spinal cord or placed at thesite such that the cells may migrate to the injury site. Alternatively,the cells are delivered intravascularly and allowed to home to the siteof injury to drive local tissue regeneration.

There are a variety of other applications where the cells may bedelivered locally to the tissue via direct injection, seeding onto amatrix for localized delivery, or delivered via the vascular systemallowing for cells to home to the site of injury or disease. Thesediseases are limb ischemia, congestive heart failure, cardiac ischemia,kidney ischemia, end stage renal disease, stroke, and ischemia of theeye.

Orthopedic Indications for Bone Regenerations

Cells have been used successfully in bone regeneration applications inhumans. Optionally, cells are mixed with 3D matrices to enhance deliveryand localization at the site where bone regeneration is required. Thethree-dimensional matrices come in a range of physical and chemicalforms, and viscous or gelled binding materials may also be added to aidhandling and delivery properties.

Three dimensional matrices include for example, demineralized boneparticles, mineralized bone particles, synthetic ceramics of the calciumphosphate family such as alpha tri-calcium phosphates (TCP), beta TCP,hydroxyappatites, and complex mixtures of these materials. Othermatrices include, for example, collagen-based sponges,polysaccharide-based materials such as hyaluronan and alginates,synthetic biodegradable polymeric materials such as poly-lactides,poly-glycolides, poly-fumarates, poly-ethylene glycol, co-polymers ofthese as well as other materials known in the art.

Any of the matrices used with cells may be processed into differentphysical forms that are common in the art for tissue regenerationapplications. These physical forms are open and closed pore foams andsponges, fiber-based woven or non-woven meshes, or small particlesranging from nano-particles to micron-sized particles (1 micrometer-1000micrometers) and macro-particles in the millimeter size scale. The smallparticles also often have an open porosity, with nanopores aiding innutrient and metabolite transport and micropores providing pores largeenough to facilitate cell seeding and tissue integration.

When the matrices used for cell delivery are small particles deliveredto wound sites, at times viscous materials or gels are used to bind theparticles that aid in materials handling and delivery, as well ashelping to keep the particles and the cells localized at the site afterplacement. Viscous binding materials include for example, hyaluronan,alginates, collagens, poly ethylene glycols, poly fumarates, blood clotsand fibrin-based clots, as well as mixtures of these materials, eitherin the form of viscous fluids to soft or hard hydrogels. Other viscousmaterials and hydrogels are known in the art

In various embodiments, cells are delivered with TCP, demineralizedbone, and mineralized bone particles in sizes ranging from 200micrometers to 5 millimeters, depending on the specific application.Optionally, these materials are bound with fibrin-based clots made fromautologous freshly prepared plasma from the patient. Other fibrin clotsor different hydrogels, or matrix materials common may also be used.

Generally, cells are mixed with the matrices just prior to surgery whenused for bone regeneration. For long-bone regeneration, typically thearea of bone non-union is opened by the surgeon, and the necrotic boneis removed. The non-unioned bone or area where bone is needed may or maynot be de-corticated by the surgeon to allow bleeding at the site, atwhich point the cell-matrix mixture is placed by the surgeon between thebones where regeneration will occur. This mixture of the cells andmatrix drive tissue regeneration with the physical matrix guiding thelocation of bone regeneration and the cells providing the tissue repairstimulus for driving angiogenesis, would healing, and bone regeneration.The remaining cell/matrix mixture is optionally placed around thefracture line after any orthopedic hardware has been placed such asplates, rods, screws or nails.

Methods of Reducing Inflammation

Inflammation is inhibited (e.g., reduced) by administering to tissue aselectin inhibitor. Tissues to be treated include any tissue subject toinflammation such as a gastrointestinal tissue, e.g., intestinal tissue,a cardiac tissue, a muscle tissue, an epithelial tissue, an endotheliumtissue, a vascular tissue, a pulmonary tissue, a dermal tissue, or ahepatic tissue. For example, the tissue is an epithelial tissue such asan intestinal epithelial tissue, pulmonary epithelial tissue, dermaltissue (i.e., skin), or liver epithelial tissue.

Inhibition of inflammation is characterized by a reduction of redness,pain and swelling of the treated tissue compared to a tissue that hasnot been contacted with a selectin inhibitor. Tissues are directlycontacted with an inhibitor. Alternatively, the inhibitor isadministered systemically. Selectin inhibitors are administered in anamount sufficient to decrease (e.g., inhibit) leukocyte-endothelialinteraction. The selectin inhibitor is administered to a subject priorto, during or after receiving G-CSF therapy. An inflammatory response isevaluated by morphologically by observing tissue damage, localizedredness, and swelling of the affected area. Alternatively, aninflammatory response is evaluated by measuring c-reactive protein, orIL-1 in the tissue or in the serum or plasma. Efficaciousness oftreatment is determined in association with any known method fordiagnosing or treating the particular inflammatory disorder. Alleviationof one or more symptoms of the inflammatory disorder indicates that thecompound confers a clinical benefit.

The methods described herein lead to a reduction in the severity or thealleviation of one or more symptoms of an inflammatory disorder. Theinflammatory disorder is acute or chronic. For example, the methodsdescribed herein reduce the severity of vascular and inflammatorycomplications associated with G-CSF therapy. Complications associatedwith G-CSF therapy include, for example, respiratory distress syndrome,angina pectoris, myocardial infarct, cutaneous leukocytoclasticvasculitis, arthritis, precipitate sickle cell vaso-occlusion, andcardiac ischemia. Disorders are diagnosed and or monitored, typically bya physician using standard methodologies.

The subject is preferably a mammal. The mammal can be, e.g., a human,non-human primate, mouse, rat, dog, cat, horse, or cow. The subjectsuffers from a disorder in which G-CSF therapy is indicated. Forexample, the subject is receiving a hematopoietic stem cell transplant.

A selectin inhibitor is a compound meant a compound that inhibits orreduces selectin-ligand interaction. Selectin inhibitors are known inthe art such as those described in U.S. Pat. No. 5,728,685 (the contentsof which are incorporated herein by reference) or are identified usingmethods described herein. The selectin inhibitor is, for example, asmall molecule, and antisense nucleic acid, a short-interfering RNA, ora ribozyme.

EXAMPLES Example 1 General Methods

Materials:

G-CSF mobilized peripheral blood (MPB) was obtained from pheresisproducts of donors for clinical HSCT at Brigham and Women'sHospital/Dana Farber Cancer Institute (Boston, Mass.). G-CSF MPBleukocytes (ML) were isolated from buffy coat of dextran sedimentatedwhole blood, followed by hypotonic lysis to remove contaminating redcells. Mononuclear fraction of these cells (ML-M) was isolated byFicoll-Hypaque (1.077 g/ml; Sigma Aldrich) density gradientcentrifugation. Native (unmobilized) peripheral blood was obtained fromconsenting healthy volunteers, and NL (buffy coat) were isolated usingdextran sedimentation as for ML. Peripheral blood mononuclear cells(PBMC) were isolated by Ficoll-Hypaque density gradient centrifugation.Polymorphonuclear leukocytes (PMN) were isolated by dextransedimentation followed by collecting the cell pellet afterFicoll-Hypaque density gradient centrifugation. Contaminated red cellswere removed by hypotonic lysis.

Normal human bone marrow (BM) cells were isolated from human BM harvestmaterial (Massachusetts General Hospital, Boston, Mass.). Red cells wereseparated using dextran sedimentation method. The leukocyte-richsupernatant was subjected to a two-step discontinuous Percoll (AmershamPharmacia Biotech; Piscataway, N.J.) density gradient centrifugation(1.065 g/ml and 1.080 g/ml; 1000 g for 20 min at 4° C.). This resultedin separation of BM cells into three different “bands” according tomyeloid cell maturity⁵⁰. The cells with least density found at the upperband (“Band 1”) contained early immature cells (myeloblasts andpromyelocytes), “Band 2” contained late immature cells (primarilymyelocytes and metamyelocytes), and “Band 3” contained the most matureleukocytes (predominantly band and segmented neutrophils) as well assome contaminating red cells, which were subsequently removed byhypotonic lysis. The cells in different bands were collected, washed andused for further studies. In some instances, BM mononuclear cells(BM-MNC) were isolated by Ficoll-Hypaque density gradientcentrifugation. CD34+/lineage− subpopulation was isolated from BM-MNCusing a negative cell selection StemSep™ human progenitor enrichmentcocktail (Stem Cell Technologies Inc.); CD34+ cells were then furtherisolated by positive selection using anti-CD34 immunomagnetic beads(Miltenyi Biotech.), routinely resulting in populations of >98% CD34+cells.

All human samples were obtained and used in accordance with theprocedures approved by the Human Experimentation and Ethics Committeesof Partners Cancer Care Institutions (Massachusetts General Hospital,Brigham and Women's Hospital and Dana Farber Cancer Institute (Boston,Mass.)).

Antibodies and E-Selectin Chimera:

The following antibodies were from BD Pharmingen (San Diego, Calif.):function blocking murine anti-human E-selectin (68-5411; IgG₁), ratanti-human CLA (HECA-452; IgM), murine anti-human PSGL-1 (KPL-1; IgG₁),purified and fluorescein isothiocynate (FITC)-conjugated murineanti-human L-selectin (DREG-56; IgG₁), murine anti-human CXCR4 (12G5;IgG_(2a)), murine anti-human CD29 (HUTS-21; IgG_(2a)), mouse IgG₁, κisotype, mouse IgG_(2a) isotype, rat IgG isotype, rat IgM isotype,FITC-conjugated goat anti-mouse Ig and FITC-conjugated goat anti-ratIgM. Rat anti-human CD44 (Hermes-1; IgG_(2a)) was a gift of Dr. BrendaSandmaier (Fred Hutchinson Cancer Research Center; Seattle, Wash.).Murine anti-human CD44 (F10-44-2; mIgM), phycoerythrin (PE)-conjugatedstrepavidin, alkaline phosphatase (AP)-conjugated anti-rat IgM, anti-ratIgG, anti-mouse Ig, and anti-human Ig were from Southern BiotechnologyAssociates (Birmingham, Ala.). Recombinant murine E-selectin/human Igchimera (E-Ig) and murine anti-human CD44 (2C5; IgG_(2a)) were from R&DSystems (Minneapolis, Minn.). Murine anti-human PSGL-1 (PL-2; IgG₁),murine anti-human CD11a (25.3, IgG₁), function blocking murineanti-human CD18 (7E4, IgG₁), murine anti-human CD49d (HP2/1, IgG₁),purified and PE-conjugated murine anti-human CD34 (QBEND10; IgG₁) andPE-conjugated mouse IgG₁, κ isotype were from Coulter-Immunotech (Miami,Fla.). Function blocking rat anti-murine E-selectin (9A9; IgG₁) was akind gift of Drs. Barry Wolitzky (CHIRON BioPharma Research; Emeryville,Calif.) and Klaus Ley (University of Virginia; Charlottesville,Va.)^(26,27).

Cell Culture and Treatment of HUVEC:

HUVEC were obtained from the tissue culture core facility at Brigham andWomen's Hospital's Pathology Department and were cultured in M199supplemented with 15% FBS, 5 units/ml heparin, 50 μg/ml endothelialgrowth factor, 100 units/ml penicillin and 100 μg/ml streptomycin. Foradhesion assays, the HUVEC were cultured at the center of 100-mm tissueculture dishes (BD Falcon; Franklin Lakes, N.J.) coated with 10 μg/mlhuman plasma fibronectin (Sigma). All experiments were performed withconfluent HUVEC monolayers. To stimulate expression of endothelialadhesion molecules including E-selectin, HUVEC were pre-treated with 20ng/ml of recombinant human TNF-α (endotoxin<0.1 ng/μg TNF-α; ResearchDiagnostics, Inc; Concord, Mass.) or 2 ng/ml of recombinant human IL-1β(endotoxin<0.1 ng/μg IL-1β; Research Diagnostics, Inc.) for 4-6 hrsprior to use in the adhesion studies.

Human hematopoietic KG1a cell line (ATCC; Manassas, Va.) was cultured asdescribed previously^(28,39). Chinese hamster ovary cells (CHO) stablytransfected with full-length cDNA encoding human E-selectin (CHO-Ecells) or human P-selectin (CHO-P cells) and mock-transfected CHO cells(CHO-mock)²⁹ were cultured in MEM supplemented with 10% FBS, 1% sodiumpyruvate, 1% non-essential amino acids, 100 units/ml penicillin and 100μg/ml streptomycin.

Parallel Plate Flow Chamber Adhesion Assays:

A tissue culture dish containing confluent HUVEC monolayer or CHO cellswas loaded into a parallel plate flow chamber (Glycotech; Gaithersburg,Md.). The flow chamber was mounted on an inverted microscope connectedto a videocamera, VCR, and monitor. The field of view was standardizedto the mid-point of the flow chamber. After a brief rinse with HBSS/10mM HEPES/2 mM CaCl₂ (assay buffer), ML or NL (1×10⁶ cells/run in assaybuffer) were drawn over the HUVEC monolayer or CHO cells at a shearstress of 1.5 dyne/cm² or 1 dyne/cm² respectively. In certainexperiments, TNF-α-stimulated HUVEC or CHO-E cells were treated withanti-human E-selectin mAb, 68-5411. The mAb treated HUVEC or CHO-E cellswere then incubated at 37° C. for 15 min prior to use in adhesionassays. In other experiments, ML were treated with anti-human L-selectinmAb, Dreg-56 or anti-human CD18 (β2 integrin) mAb, 7E4 or anti-humanCD29 (β1 integrin) mAb, HUTS-21 or mouse IgG_(1,k) at 4° C. for 15 minprior to use in adhesion assays. Primary tethering⁴⁹ was determined byquantifying the number of ML or NL that attached (i.e., interacted) fromthe free stream directly onto HUVEC monolayer or CHO cells during thefirst 2 minutes of flow. Secondary attachments (i.e., flowing leukocytesinteracting with an already adherent leukocyte on HUVEC monolayer or CHOcells) or leukocytes that rolled into the field of observation from theupstream region were not counted. Average rolling velocity, aquantitative measure of selectin binding strength, was computed (usingScion Image) as the displacement by the centroid of the cell divided bythe time interval of observation, 5 sec.

In Vivo Imaging of Cellular Trafficking:

All studies were performed in accordance with NIH guidelines for thecare and use of animals and under approval of the Institutional AnimalCare and Use Committees of Partners Affiliated Institutions and theHarvard Medical School. Groups of C57BL/6 mice received intradermalinjection of 200 ng/ml of TNF-α in a volume of 10 μl into the right ear.As a control, the left ear pinnae received intradermal injection of PBS.Six hours later, an intravenous catheter was inserted into the tail veinof anesthetized mice. The anesthetized mice were placed in a heated tubeon the stage of a video rate scanning laser confocal microscopeplatform²⁵. To image the vasculature, the ears of mice were placed on acoverslip and high-resolution images with cellular details was obtainedthrough the intact mouse skin at depths of up to 250 μm from the surfaceusing a Olympus 60×1.2NA water immersion objective lens. For celltracking, animals received 5×10⁶ of DiD labeled (Molecular Probes) ML orNL suspended in 200 μl sterile saline (tail-vein injection), while onthe stage, and their interaction with ear vasculature was viewed usingin vivo confocal microscopy. DiD was excited with a 656 nm diode laserand detected with a photomultiplier tube through a 695+/−27.5 nmbandpass filter (Omega Optical). The system operates at auser-selectable frame rate from 15-30 fps. The images are simultaneouslyrecorded by a digital video recorder (Canon) and captured by a Macintoshcomputer equipped with a Scion LG-3 board for frame averaging²⁵. In someexperiments, mice were treated with 70 μg of mAb 9A9 for 1 hr. prior toinjection of leukocytes. Average rolling velocity was computed (usingImageJ) as the displacement by the centroid of the cell divided by thetime interval of observation.

OSGE Treatment:

ML (10×10⁶ cells/ml) were treated at 37° C. for 1 hr with 30 μg/ml OSGE(Cedarlane Laboratories, Ontario, Canada). Following the incubation, thecells were washed and divided proportionately for use in flow cytometry,Western blot analysis, and parallel plate flow chamber adhesion assays.

G-CSF Treatment:

For in vitro G-CSF treatment, isolated subsets of BM cells (1×10⁶cells/ml) were cultured at 37° C. for 72 hr in the presence ofrecombinant human G-CSF (10 ng/ml in RPMI, 10% FBS; Amgen, ThousandOaks, Calif.)); PBS diluent was used for control (untreated) cells. Notethat the in vitro dose of G-CSF utilized is well within the expectedlevels in human serum/extracellular fluids (after a single subcutaneousdose of 5 or 10 μg/kg, peak serum levels range from ˜15.1 to ˜100.5ng/ml)^(44,45). In all experiments, L-selectin expression was determinedby flow cytometry on untreated and G-CSF-treated cells to test theefficacy of G-CSF treatment. At the end of the culture period, equalnumbers of untreated and G-CSF treated cells were dividedproportionately for use in flow cytometry, Western blot analysis, RT-PCRand parallel plate flow chamber adhesion assays. For Western blotanalysis, cells were lysed in 2% NP-40 in Buffer A (consisting of 150 mMNaCl, 50 mM Tris-HCl pH 7.4, 1 mM EDTA, 0.02% sodium azide, 20 mg/mlPMSF and 1 complete protease inhibitor cocktail tablet/100 ml buffer).The lysate was used immediately or stored at −20° C. for later use.

Membrane Preparations, SDS Separation and Western Blots:

Membrane proteins of purified ML-M, ML-G, PMN, or PBMC were isolated asdescribed previously^(28,39). Membrane protein suspensions werealiquoted and stored at −20° C. For SDS-PAGE and Western blotting,membrane preparations were diluted in reducing sample buffer, boiled andthen separated on 4-20% or 7.5% Criterion Tris-HCl SDS-PAGE gels(Bio-Rad Laboratories). Resolved membrane proteins were transferred toSequi-blot polyvinylidene diflouride (PVDF) membrane (Bio-RadLaboratories) and blocked with heat inactivated FBS. Blots wereincubated with primary antibodies or E-Ig (each at 1 μg/ml). Appropriateisotype control immunoblots (each at 1 μg/ml) were performed in parallelto evaluate nonspecific binding to protein bands. After extensivewashing with TBS/0.1% Tween 20, blots were incubated with appropriatealkaline phosphatase (AP)-conjugated secondary antibodies (1:1000).Western Blue AP substrate (Promega, Madison, Wis.) was used to developthe blots.

Immunoprecipitation Studies:

Membrane proteins of ML-M were incubated with immunoprecipitatingantibodies or E-Ig, or with appropriate isotype controls and thenincubated with Protein G-agarose. Immunoprecipitates were washedextensively using Buffer A containing 2% NP-40, 1% SDS. E-Igprecipitated material was washed extensively with Buffer A without EDTAcontaining 2% NP-40 and 2 mM CaCl₂. All immunoprecipitates were dilutedin reducing sample buffer, boiled, then subjected to SDS-PAGE,transferred to PVDF membrane, and immunostained with HECA-452 orappropriate mAbs.

In some experiments, the surface proteins on ML-M and KG1a cells werebiotinylated using EZ-Link® NHS-PEO₄-Biotin (Pierce Biotechnology, Inc.;Rockford, Ill.). An aliquot of cells (1×10⁶ cells) was removed and theefficiency of biotinylation determined using flow cytometry. Theremaining cells were solubilized in Buffer A containing 2% NP-40.L-selectin was immunoprecipitated using polyclonal rabbit anti-humanL-selectin antiserum (prepared by Covance, Princeton, N.J.). Controlimmunoprecipitation was performed in parallel using rabbit pre-immuneserum (Covance). Immunoprecipitated proteins were diluted in reducingsample buffer, boiled, then subjected to SDS-PAGE, transferred to PVDFmembrane and immunostained with horseradish peroxidase (HRP) conjugatedstrepavidin (1:500; Dako Cytomation; Carpinteria, Calif.). Vector NovaRed HRP substrate (Vector Laboratories; Burlingame, Calif.) was used todevelop the blots.

Blot Rolling Assays:

The blot rolling assay has been described previously²⁹. CHO-E werewashed in PBS, and resuspended to 2×10⁶ cells/ml in assay buffer/10%glycerol. Western blots of ML-M membrane preparations stained withHECA-452 were rendered translucent by immersion in assay buffer/10%glycerol. These blots were then placed in the parallel plate flowchamber, and CHO-E were perfused into the chamber at a shear stress of0.6 dyne/cm²; an adjustment in the volumetric flow rate was made toaccount for the increase in viscosity due to the presence of 10%glycerol in the assay buffer. Molecular weight standards (SeeBlue® Plus2prestained molecular weight standard; Invitrogen Corporation; Carlsbad,Calif.) were co-electrophoresed on adjacent lanes and served as a guideto aid placement of the flow chamber over the stained bands of interest.The number of tethering and rolling CHO-E was tabulated as function ofthe molecular weight region and compiled into an adhesion histogram.Negative controls were prepared by adding 10 mM EDTA to the assay bufferto chelate Ca²⁺ required for binding or treating CHO-E with antiE-selectin mAb, 68-5411, at 4° C. for 15 min. prior to use in adhesionassays.

Flow Cytometry:

Aliquots of ˜2−5×10⁵ cells were washed with PBS, 2% FBS and incubatedwith primary mAbs. Subsequently, the cells were washed and incubatedwith species and isotype matched FITC- or PE-labeled polyclonalantibodies. Following this incubation, the cells were washed and FITC orPE fluorescence of cells was determined using a Cytomics FC 500 MPL flowcytometer (Beckman Coulter Inc., Fullerton, Calif.).

RT-PCR:

Total cellular RNA was isolated from equal numbers of NL and ML oruntreated or G-CSF-treated human BM cells using Trizol® LS reagent (LifeTechnologies, Inc.) according to the manufacturer's protocol. Theisolated RNA was quantified by spectrophotometric absorbance readings at260 nm. Equal amounts of RNA were then taken and used as templates forRT-PCR with Titan™ One Tube RT-PCR System (Roche Molecular Biochemicals)and the following primers: ST3Gal IV, sense CTC TCC GAT ATC TGT TTT ATTTTC CCA TCC CAG AGA GAA GAA GGA G (SEQ ID NO:1) and antisense GAT TAAGGT ACC AGG TCA GAA GGA GGT GAG GTT CTT (SEQ ID NO:2); FucT-VII, senseCCC ACC GTG GCC CAG TAC TAC CGC TTC T (SEQ ID NO:3) and antisense CTGACC TCT GTG CCC AGC CTC CCG T (SEQ ID NO:4); FucT-IV, sense CGG GTG TGCCAG GCT GTA CAG AGG (SEQ ID NO:5) and antisense TCG GGA ACA GTT GTG TATGAG ATT (SEQ ID NO:6); GAPDH sense GAA GGT GAA GGT CGG AGT C (SEQ IDNO:8) and antisense GAA GAT GGT GAT GGG ATT TC (SEQ ID NO:9).

A total of 30 cycles were found to be below the plateau phase ofamplification for all primers giving an accurate reflection of therelative concentration of mRNA. Optical PCR conditions were 94° C. for 2min, 60° C. for 45 sec., and 72° C. for 1 min on a PTC-200 PeltierThermal cycler (MJ Research). Amplified bands were visualized after 1%agarose (Sigma Aldrich) gel electrophoresis of the PCR products.Analysis of digital images of amplified bands was done using Kodaksoftware. Mean intensities were determined of fixed size regions setover each band. The background intensity for each lane was subtractedfrom mean intensity in the same lane to arrive at net intensity. The netintensity of the specific band was then normalized to the net intensityof GAPDH control.

Statistics:

When comparing two means, statistical analyses were done by unpairedStudent's t-test of the means. P values<0.05 were consideredstatistically significant. Unless stated otherwise, all error barsrepresent standard error of mean.

Example 2 ML Possess Enhanced Binding to E-Selectin Relative to NL

We analyzed adhesive interactions of ML and NL on TNF-α-stimulated humanumbilical vein endothelial cells (HUVEC) in a parallel plate flowchamber assay. Under hemodynamic flow conditions (1.5 dyne/cm²), MLdisplayed markedly enhanced E-selectin-mediated and Ca²⁺-dependentprimary tethering on stimulated HUVEC compared to NL (FIG. 1 a).Moreover, ML rolled distinctly slower than NL on stimulated HUVEC (FIG.1 b). Because activated integrins support deceleration of cells in flow,we measured the surface expression of activation-dependent epitopes ofintegrins LFA-1 (CD11a/CD18;α_(L)β₂) and VLA-4 (CD49d/CD29;α₄β₁), and ofchemokine-receptor CXCR4 on ML and NL. Flow cytometry revealed nodifference in the expression of these molecules (FIG. 7 a) suggestingthat the marked decrease in rolling velocity of ML was primarily due totheir increased capacity to engage endothelial E-selectin. To furthercharacterize the enhanced E-selectin binding capacity of ML, we examinedthe adhesion of ML and NL on Chinese hamster ovary cells transfectedwith human E-selectin (CHO-E). ML displayed significantly enhancedE-selectin-mediated and Ca²⁺-dependent primary tethering on CHO-E (FIG.1 c). Furthermore, the rolling velocity of ML on CHO-E was significantlylower than that of NL (FIG. 1 d).

To assess whether the observed enhanced E-selectin binding of ML invitro could have a meaningful physiologic effect in vivo, we utilizedTNF-α-induced murine ear inflammation model and employed dynamicreal-time intravital confocal microscopy²⁵ to visualize the adhesiveinteractions of ML and NL with inflamed ear vasculature. Compared to NL,ML displayed significantly slower rolling (FIG. 1 e) and significantlyenhanced adhesion (FIGS. 1 f and 1 g) to vascular endothelium within theTNF-α-treated ear. A function blocking mAb to murine E-selectin,9A9^(26,27), prominently increased the rolling velocity and diminishedleukocyte adhesion to inflamed endothelium, highlighting a critical rolefor vascular E-selectin/leukocyte E-selectin ligand interactions inmediating this enhanced adhesion. Collectively, these data demonstratethat ML possess heightened adhesive interactions with endotheliummediated by enhanced binding to E-selectin.

Example 3 ML Express Hcell and a Novel HECA-452-Reactive ˜65 kDaE-Selectin Glycoprotein Ligand

To identify the E-selectin ligand(s) expressed by ML, we performedWestern blot analysis using mAb HECA-452 as a probe. This mAb HECA-452recognizes sialofucosylated oligosaccharides, prototypically sialyllewis-X (sLe^(x)), that serve as selectin binding determinants andHECA-452 reactivity of glycoproteins correlates with E-selectin ligandactivity^(28,29). Western-blot analysis of unfractionated ML lysatesrevealed several sialidase-sensitive HECA-452-reactive bands (˜220 kDa,˜130 kDa, ˜100 kDa, and ˜65 kDa) (FIG. 2 a and FIG. 8). Consistent withresults of prior studies³⁰, unfractionated NL lysates revealed twoprominent HECA-452-reactive bands (˜220 kDa and ˜130 kDa) (FIG. 2 a).Notably, comparison of ML to NL showed no significant difference inHECA-452 staining of bands at ˜220 kDa and ˜130 kDa; however, ML lysatesshowed distinct and prominent HECA-452 staining at bands of ˜100 kDa and˜65 kDa (FIG. 2 a). Western blot analysis of membrane proteins ofmononuclear (ML-M) and polymorphonuclear (ML-G) fractions of ML eachshowed the marked expression of HECA-452-reactive species of ˜100 kDaand ˜65 kDa (FIG. 2 b).

To determine whether the HECA-452-reactive membrane glycoproteins fromML represented E-selectin ligands, we utilized the blot-rollingassay^(29,31). For these experiments, we could not assess bindinginteractions over the entire lane due to the length restriction of theflow chamber; because the differences in HECA-reactivity in proteinsfrom ML and NL clustered within mobilities encompassing 30 kDa-170 kDa,we set the field view over this range. Among ML lysates, E-selectinligand activity was reproducibly observed on HECA-452 stained bands at˜130 kDa, ˜100 kDa and ˜65 kDa (FIG. 2 c). Notably, the number ofinteracting CHO-E was much greater on the ˜100 kDa and ˜65 kDa bandscompared to the ˜130 kDa band (FIG. 2 c), suggesting that theseglycoproteins were major E-selectin ligands. Specificity for E-selectinbinding was verified by significant diminution of CHO-E binding byaddition of EDTA to cell suspension or by incubating cells with functionblocking mAb to E-selectin (not shown). Moreover, no interactions wereobserved when mock-transfected CHO cells were perfused over HECA-452blots of ML (not shown).

In a complementary approach, we probed the expression of E-selectinligands using murine E-selectin-human Ig chimera (E-Ig) in ligand blots.E-Ig, in the presence of Ca²⁺, stained ML-M membrane proteins at ˜220kDa, ˜130 kDa, ˜100 kDa, and ˜65 kDa (FIG. 2 d) corresponding with theHECA-452-reactive membrane glycoproteins, whereas these bands did notstain in the presence of EDTA or with control human-Ig (FIG. 9).HECA-452 blots of membrane proteins of ML-M immunoprecipitated usingE-Ig also revealed staining at ˜220 kDa, ˜130 kDa, ˜100 kDa and ˜65 kDa(FIG. 2 e). Control immunoprecipitates using human-Ig or E-Ig in thepresence of EDTA showed absence of HECA-452-reactive proteins (FIG. 2 e,and not shown). Collectively, these results show that the observedglycoproteins migrating at ˜220 kDa, ˜130 kDa, ˜100 kDa and ˜65 kDarepresent the E-selectin ligands of ML.

The electrophoretic mobilities of several bands bearing E-selectinligand activity coincided with that of two previously characterizedhuman E-selectin ligands, i.e., PSGL-1 (mw 220-240 kDa (dimer) and120-140 kDa (monomer)) and the Hcell glycoform of CD44 (mw ˜100kDa)^(28,31). Thus, we sought to determine whether these bandsrepresented these molecules. KPL-1 (anti-PSGL-1) blots of ML-M membraneproteins showed bands of ˜220 kDa and ˜130 kDa under reducing conditions(FIG. 3 a). Subsequently, blots of immunoprecipitated PSGL-1 werestained with either HECA-452 or KPL-1. As shown in FIG. 3 b, ML expressboth the monomer and dimer forms of PSGL-1, which represent the ˜130 kDaand ˜220 kDa HECA-452-reactive glycoproteins. Blots of ML-M membranepreparations stained with Hermes-1 (anti-CD44) showed a band of ˜100 kDaunder reducing conditions (FIG. 3 c). Subsequently, blots ofHermes-1-immunoprecipitated CD44 were stained with either HECA-452 or2C5, another anti-human CD44 antibody. As shown in FIG. 3 d, ML expressHcell, evident as a HECA-452-reactive glycoform of CD44²⁸.

The HECA-452-reactive ˜65 kDa glycoprotein was not immunoprecipitated bymAbs KPL-1 or Hermes-1 (FIGS. 3 b and 3 d). Other mAbs to PSGL-1 (e.g.,PL-2) and CD44 (e.g., 2C5) also did not immunoprecipitate the ˜65 kDaprotein (FIG. 10), suggesting that this protein is not related to eitherPSGL-1 or CD44. Human neutrophil L-selectin (mw ˜75-90 kDa) has beenreported to be an E-selectin ligand³². Since the HECA-452-reactive ˜65kDa glycoprotein resolved in Western blots in the molecular weight rangeof L-selectin³³, we investigated whether this structure was L-selectin.Flow cytometry revealed that ML express little L-selectin (FIG. 3 e).This observation is consistent with a previous study demonstrating thatG-CSF treatment of leukocytes down-regulates L-selectin expression³⁴. Inagreement with the flow cytometry results, we were unable to detectL-selectin in immunoprecipitates of ML-M (FIG. 3 f). Collectively, thesedata demonstrate that PSGL-1 and Hcell serve as E-selectin ligands on MLand that the HECA-452-reactive ˜65 kDa protein is not a glycoform ofPSGL-1, CD44 or L-selectin.

Example 4 ML Possess Enhanced Levels of ST3GalIV, FucT-IV and FucT-VII

The capacity of E-selectin to recognize its relevant glycoproteinleukocyte ligand(s) is dependent on carbohydrate decoration of the coreprotein^(18,28,35). Based on prior observations thatglycosyltransferases are regulated by cytokines³⁶, we sought toinvestigate whether G-CSF affects expression of relevantglycosyltransferases that create pertinent sialofucosylations. Thecarbohydrate modifications rendering the expression of E-selectinbinding determinants are critically mediated by specificglycosyltransferases: α2,3-sialyltransferase (ST3GalIV) and leukocyteα1,3-fucosyltransferases (FucT-IV and FucT-VII)^(37,38). RT-PCR analysisof the expression of ST3GalIV, FucT-IV and FucT-VII revealed that thetranscripts for each of these glycosyltransferases were increased in MLrelative to NL (FIG. 4).

Example 5 Hcell and ˜65 kDa Glycoprotein are Major E-Selectin Ligands onML

The E-/L-selectin ligand activity of Hcell is resistant toO-sialoglycoprotein endopeptidase (OSGE) treatment^(28,31,39), whereasOSGE treatment abrogates PSGL-1 binding to all three selectins^(40,41).Accordingly, to determine the contribution of PSGL-1 to the observedenhanced E-selectin ligand activity of ML, we performed OSGE digestionand measured residual E-selectin binding activity. OSGE digestion of MLabrogated surface expression of PSGL-1 and had minimal effect on CD44and HECA-452 antigen levels (FIG. 5 a). E-Ig blots of cell lysates of MLshowed distinct reduction of E-selectin binding by PSGL-1 onOSGE-treated cells (absent binding at dimer and marked diminution atmonomer), while E-selectin binding determinants of Hcell and the ˜65 kDaglycoprotein were intact (FIG. 5 b). Despite significant decreases inPSGL-1 expression and function, OSGE treatment had no effect on primarytethering of ML on TNF-α-stimulated HUVEC (FIG. 5 c). Combined, thesedata demonstrate that Hcell and the ˜65 kDa glycoprotein are majorE-selectin ligands on ML. Interestingly, compared to NL, ML displayedmarkedly diminished P-selectin-mediated and Ca²⁺-dependent primarytethering on Chinese hamster ovary cells transfected with humanP-selectin (CHO-P) (FIG. 11 a). Furthermore, ML rolled significantlyfaster than NL on CHO-P (FIG. 11 b). Given that PSGL-1 is thepredominant ligand for P-selectin, the decreased PSGL-1 function on ML,combined with the findings that G-CSF treatment down-regulates PSGL-1expression in humans⁴², indicates that Hcell and ˜65 kDa glycoproteincontribute to the augmented E-selectin binding of ML.

Example 6 In Vitro G-CSF Treatment of Human BM Cells Up-Regulates theExpression of ST3GalIV, FucT-IV and FucT-VII with Associated Increasesin Expression of Hcell and HECA-452-Reactive ˜65 kDa Glycoprotein andE-Selectin Binding

While PSGL-1 is broadly expressed on myeloid, lymphoid and dendriticlineage cells^(30,43) and also on CD34+ hematopoietic progenitor cells(FIG. 6 a), Hcell is typically expressed only on CD34+ progenitors inhuman BM (FIG. 6 a and²⁸). Separation of ML into early immature (Band1), late immature (Band 2) and mature (Band 3) myeloid fractions byPercoll gradient showed that predominantly Band 1 and Band 2 cellspossessed Hcell and also expressed the HECA-452-reactive ˜65 kDaglycoprotein (FIG. 6 b). The presence of these structures in associationwith G-CSF administration prompted us to determine whether G-CSFdirectly induces their expression. Freshly isolated BM cells wereseparated into early immature (Band 1), late immature (Band 2) andmature (Band 3) myeloid fractions. The different subsets of cells werethen cultured at 37° C. for 72 hr in the presence of 10 ng/ml G-CSF.Importantly, the in vitro dose of G-CSF utilized is well within theexpected levels in human serum/extracellular fluids (after a singlesubcutaneous dose of 5 or 10 μg/kg, peak serum levels range from ˜15.1to ˜100.5 ng/ml)^(44,45). The expression of Hcell and ˜65 kDaglycoprotein were analyzed by reactivity to HECA-452 and E-Ig usingWestern blot analysis. In all experiments, efficacy of G-CSF treatmentwas confirmed by observing the down-regulation of L-selectin expressionon G-CSF treated cells relative to untreated cells (not shown). As shownin FIG. 6 c, G-CSF treatment consistently resulted in up-regulation inexpression of Hcell and the HECA-452-reactive ˜65 kDa glycoproteinpredominantly on immature Band 1 and Band 2 cells. In association withG-CSF-induced increases in HCELL and HECA-452-reactive ˜65 kDa protein,in vitro treatment of immature human BM cells (Band 2) with G-CSFresulted in increases in expression levels of ST3GalIV, FucT-IV andFucT-VII (FIGS. 6 d and 6 e) together with enhanced E-selectin binding(FIG. 12).

Example 7 In Vitro G-CSF Treatment of Human BM Cells and Blood CellsUp-Regulates the Expression of Fucosyltransferase IX (FuT-IX) withAssociated Increases in Expression of CD15

Freshly isolated bone marrow and peripheral blood were t cultured at 37°C. for 72 hr in the presence of 10 ng/ml G-CSF. Treatment increasesFuct-IX expression in normal progenitors and in mobilized peripheralblood. (FIG. 13) Fuct-IX has been found to regulate CD15 expression(which is the non-sialylated core of sLex) and is expressed on thesurface of neutrophils. CD15 plays a role in innate immunity in primingdendritic cells. Moreover, FTIX fucosylates internal lactosamine unitsyielding the VIM-2 epitope. While, the VIM-2 is not sLex, but itconsists of a terminal sialic acid with an “internal fucosylation” thatcan function as an E-selectin ligand

Example 8 In Vitro G-CSF Treatment of Human BM Cells and LeukocytesCells Up-Regulates the Expression of Sialidase with Associated Increasesin Expression of CD15

Freshly isolated BM cells were separated into early immature (Band 1),late immature (Band 2) and mature (Band 3) myeloid fractions. Earlyimmature bone marrow cells, late mature bone marrow cells and leukocyteswere then cultured at 37° C. for 72 hr in the presence of 10 ng/mlG-CSF. Treatment of bone marrow and leukocytes resulted in an increaseexpression of sialidase. Treatment of early immature none marrow did nothas an effect on the expression of CD15. However treatment of the latemature bone marrow and leukocytes resulted in an increased expression ofCD15. Furthermore, this increase of CD15 could be prevented by theinhibition of sialidase with 2-deoxy-2,3-dehydro-N-acetyl-neuraminicacid (DANA). These results indicate that CD15 expression was a directresult of the increase of sialidase expression resulting from G-CSFtreatment.

REFERENCES

-   1. Elfenbein, G. J. & Sackstein, R. Primed marrow for autologous and    allogeneic transplantation: a review comparing primed marrow to    mobilized blood and steady-state marrow. Exp Hematol 32, 327-39    (2004).-   2. Lindemann, A. & Rumberger, B. Vascular complications in patients    treated with granulocyte colony-stimulating factor (G-CSF). Eur J    Cancer 29A, 2338-9 (1993).-   3. Hill, J. M. & Bartunek, J. The end of granulocyte    colony-stimulating factor in acute myocardial infarction? Reaping    the benefits beyond cytokine mobilization. Circulation 113, 1926-8    (2006).-   4. Azoulay, E., Attalah, H., Harf, A., Schlemmer, B. & Delclaux, C.    Granulocyte colony-stimulating factor or neutrophil-induced    pulmonary toxicity: myth or reality? Systematic review of clinical    case reports and experimental data. Chest 120, 1695-701 (2001).-   5. Azoulay, E. et al. Exacerbation by granulocyte colony-stimulating    factor of prior acute lung injury: implication of neutrophils. Crit.    Care Med 30, 2115-22 (2002).-   6. Arimura, K. et al. Acute lung Injury in a healthy donor during    mobilization of peripheral blood stem cells using granulocyte-colony    stimulating factor alone. Haematologica 90, ECR10 (2005).-   7. Fukumoto, Y. et al. Angina pectoris occurring during granulocyte    colony-stimulating factor-combined preparatory regimen for    autologous peripheral blood stem cell transplantation in a patient    with acute myelogenous leukaemia. Br J Haematol 97, 666-8 (1997).-   8. Mossner, R., Beckmann, I., Hallermann, C., Neumann, C. &    Reich, K. Granulocyte colony-stimulating-factor-induced psoriasiform    dermatitis resembles psoriasis with regard to abnormal cytokine    expression and epidermal activation. Exp Dermatol 13, 340-6 (2004).-   9. Dereure, O., Hillaire-Buys, D. & Guilhou, J. J.    Neutrophil-dependent cutaneous side-effects of leucocyte    colony-stimulating factors: manifestations of a neutrophil recovery    syndrome? Br J Dermatol 150, 1228-30 (2004).-   10. Jain, K. K. Cutaneous vasculitis associated with granulocyte    colony-stimulating factor. J Am Acad Dermatol 31, 213-5 (1994).-   11. Stricker, R. B. & Goldberg, B. G-CSF and exacerbation of    rheumatoid arthritis. Am J Med 100, 665-6 (1996).-   12. Adler, B. K. et al. Fatal sickle cell crisis after granulocyte    colony-stimulating factor administration. Blood 97, 3313-4 (2001).-   13. Hill, J. M. et al. Outcomes and risks of granulocyte    colony-stimulating factor in patients with coronary artery disease.    J Am Coll Cardiol 46, 1643-8 (2005).-   14. Harada, M. et al. G-CSF prevents cardiac remodeling after    myocardial infarction by activating the Jak-Stat pathway in    cardiomyocytes. Nat Med 11, 305-11 (2005).-   15. Butcher, E. C. Leukocyte-endothelial cell recognition: three (or    more) steps to specificity and diversity. Cell 67, 1033-6 (1991).-   16. Sackstein, R. The lymphocyte homing receptors: gatekeepers of    the multistep paradigm. Curr Opin Hematol 12, 444-50 (2005).-   17. van der Wal, A. C., Das, P. K., Tigges, A. J. & Becker, A. E.    Adhesion molecules on the endothelium and mononuclear cells in human    atherosclerotic lesions. Am J Pathol 141, 1427-33 (1992).-   18. Kansas, G. S. Selectins and their ligands: current concepts and    controversies. Blood 88, 3259-87 (1996).-   19. Albert, R. K. Mechanisms of the adult respiratory distress    syndrome: selectins. Thorax 50 Suppl 1, S49-52 (1995).-   20. Kriegsmann, J. et al. Expression of E-selectin messenger RNA and    protein in rheumatoid arthritis. Arthritis Rheum 38, 750-4 (1995).-   21. Groves, R. W., Allen, M. H., Barker, J. N., Haskard, D. O. &    MacDonald, D. M. Endothelial leucocyte adhesion molecule-1 (ELAM-1)    expression in cutaneous inflammation. Br J Dermatol 124, 117-23    (1991).-   22. Glass, L. F., Fotopoulos, T. & Messina, J. L. A generalized    cutaneous reaction induced by granulocyte colony-stimulating factor.    J Am Acad Dermatol 34, 455-9 (1996).-   23. Bussolino, F. et al. Granulocyte- and    granulocyte-macrophage-colony stimulating factors induce human    endothelial cells to migrate and proliferate. Nature 337, 471-3    (1989).-   24. Fuste, B. et al. Granulocyte colony-stimulating factor increases    expression of adhesion receptors on endothelial cells through    activation of p38 MAPK. Haematologica 89, 578-85 (2004).-   25. Sipkins, D. A. et al. In vivo imaging of specialized bone marrow    endothelial microdomains for tumour engraftment. Nature 435, 969-73    (2005).-   26. Smith, M. L., Olson, T. S. & Ley, K. CXCR2- and    E-selectin-induced neutrophil arrest during inflammation in vivo. J    Exp Med 200, 935-9 (2004).-   27. Kunkel, E. J. & Ley, K. Distinct phenotype of    E-selectin-deficient mice. E-selectin is required for slow leukocyte    rolling in vivo. Circ Res 79, 1196-204 (1996).-   28. Dimitroff, C. J., Lee, J. Y., Rafii, S., Fuhlbrigge, R. C. &    Sackstein, R. CD44 is a major E-selectin ligand on human    hematopoietic progenitor cells. J Cell Biol 153, 1277-86 (2001).-   29. Fuhlbrigge, R. C., King, S. L., Dimitroff, C. J., Kupper, T. S.    & Sackstein, R. Direct real-time observation of E- and    P-selectin-mediated rolling on cutaneous lymphocyte-associated    antigen immobilized on Western blots. J Immunol 168, 5645-51 (2002).-   30. Kieffer, J. D. et al. Neutrophils, monocytes, and dendritic    cells express the same specialized form of PSGL-1 as do skin-homing    memory T cells: cutaneous lymphocyte antigen. Biochem Biophys Res    Commun 285, 577-87 (2001).-   31. Dimitroff, C. J., Lee, J. Y., Fuhlbrigge, R. C. & Sackstein, R.    A distinct glycoform of CD44 is an L-selectin ligand on human    hematopoietic cells. Proc Natl Acad Sci USA 97, 13841-6 (2000).-   32. Zollner, O. et al. L-selectin from human, but not from mouse    neutrophils binds directly to E-selectin. J Cell Biol 136, 707-16    (1997).-   33. Schleiffenbaum, B., Spertini, O. & Tedder, T. F. Soluble    L-selectin is present in human plasma at high levels and retains    functional activity. J Cell Biol 119, 229-38 (1992).-   34. Ohsaka, A. et al. Granulocyte colony-stimulating factor    down-regulates the surface expression of the human leucocyte    adhesion molecule-1 on human neutrophils in vitro and in vivo. Br J    Haematol 84, 574-80 (1993).-   35. Fuhlbrigge, R. C., Kieffer, J. D., Armerding, D. & Kupper, T. S.    Cutaneous lymphocyte antigen is a specialized form of PSGL-1    expressed on skin-homing T cells. Nature 389, 978-81 (1997).-   36. Wagers, A. J., Waters, C. M., Stoolman, L. M. & Kansas, G. S.    Interleukin 12 and interleukin 4 control T cell adhesion to    endothelial selectins through opposite effects on alpha    1,3-fucosyltransferase VII gene expression. J Exp Med 188, 2225-31    (1998).-   37. Ellies, L. G. et al. Sialyltransferase specificity in selectin    ligand formation. Blood 100, 3618-25 (2002).-   38. Wagers, A. J., Stoolman, L. M., Kannagi, R., Craig, R. &    Kansas, G. S. Expression of leukocyte fucosyltransferases regulates    binding to E-selectin: relationship to previously implicated    carbohydrate epitopes. J Immunol 159, 1917-29 (1997).-   39. Dimitroff, C. J., Lee, J. Y., Schor, K. S., Sandmaier, B. M. &    Sackstein, R. Differential L-selectin binding activities of human    hematopoietic cell L-selectin ligands, Hcell and PSGL-1. J Biol Chem    276, 47623-31 (2001).-   40. Goetz, D. J. et al. Isolated P-selectin glycoprotein ligand-1    dynamic adhesion to P- and E-selectin. J Cell Biol 137, 509-19    (1997).-   41. Spertini, O., Cordey, A. S., Monai, N., Giuffre, L. &    Schapira, M. P-selectin glycoprotein ligand 1 is a ligand for    L-selectin on neutrophils, monocytes, and CD34+ hematopoietic    progenitor cells. J Cell Biol 135, 523-31 (1996).-   42. Jilma, B. et al. Rapid down modulation of P-selectin    glycoprotein ligand-1 (PSGL-1, CD162) by G-CSF in humans.    Transfusion 42, 328-33 (2002).-   43. Laszik, Z. et al. P-selectin glycoprotein ligand-1 is broadly    expressed in cells of myeloid, lymphoid, and dendritic lineage and    in some nonhematopoietic cells. Blood 88, 3010-21 (1996).-   44. van Der Auwera, P. et al. Pharmacodynamics and pharmacokinetics    of single doses of subcutaneous pegylated human G-CSF mutant (Ro    25-8315) in healthy volunteers: comparison with single and multiple    daily doses of filgrastim. Am J Hematol 66, 245-51 (2001).-   45. Faulkner, L. B. et al. G-CSF serum pharmacokinetics during    peripheral blood progenitor cell mobilization: neutrophil    count-adjusted dosage might potentially improve mobilization and be    more cost-effective. Bone Marrow Transplant 21, 1091-5 (1998).-   46. Hakans son, L. et al. Effects of in vivo administration of G-CSF    on neutrophil and eosinophil adhesion. Br J Haematol 98, 603-11    (1997).-   47. Xia, L. et al. P-selectin glycoprotein ligand-1-deficient mice    have impaired leukocyte tethering to E-selectin under flow. J Clin    Invest 109, 939-50 (2002).-   48. Yang, J. et al. Targeted gene disruption demonstrates that    P-selectin glycoprotein ligand 1 (PSGL-1) is required for    P-selectin-mediated but not E-selectin-mediated neutrophil rolling    and migration. J Exp Med 190, 1769-82 (1999).-   49. Zou, X. et al. PSGL-1 Derived from Human Neutrophils is a High    Efficiency Ligand for Endothelial Expressed E-selectin under Flow.    Am J Physiol Cell Physiol (2005).-   50. Cowland, J. B. & Borregaard, N. Isolation of neutrophil    precursors from bone marrow for biochemical and transcriptional    analysis. J Immunol Methods 232, 191-200 (1999).

OTHER EMBODIMENTS

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

What is claimed is:
 1. A method for increasing cell surface expressionor activity of an E-selectin ligand on a myeloid cell comprising,contacting the myeloid cell with granulocyte colony stimulating factor(G-CSF) and a sialidase inhibitor, thereby increasing cell surfaceexpression or activity of an E-selectin ligand on the cell.
 2. Themethod of claim 1, wherein a plurality of myeloid cells are provided. 3.The method of claim 1, wherein the myeloid cell is autologous.
 4. Themethod of claim 1, wherein the myeloid cell is allogeneic.
 5. The methodof claim 1, wherein the myeloid cell is provided ex vivo.
 6. The methodof claim 1, wherein the myeloid cell is contacted with G-CSF in vitro.7. The method of claim 1, wherein the E-selectin ligand is ahematopoietic cell E-/L-selectin Ligand (HCELL) polypeptide.
 8. Themethod of claim 1, wherein said method increases the expression of aHECA-452-reactive epitope on the cell.
 9. The method of claim 1, whereinthe cell is treated ex vivo, and wherein the method further comprisesadministering the cell to a subject in need thereof.
 10. The method ofclaim 8, wherein the cell is administered to a subject as part oftreatment with hematopoietic stem cell transplantation.
 11. The methodof claim 8, wherein the cell is administered to a subject who is in needof treatment for tissue injury.
 12. The method of claim 1, wherein thesialidase inhibitor is 2-deoxy-2,3-dehydro-N-acetyl-neuraminic acid(DANA).
 13. The method of claim 1, wherein the cell is administered to asubject as part of treatment with hematopoietic stem celltransplantation.
 14. The method of claim 1, wherein the cell isadministered to a subject as part of treatment for infection.
 15. Themethod of claim 1, wherein the cell is administered to a subject who isin need of treatment for tissue injury.
 16. The method of claim 1,wherein the cell is a native human myeloid cell.
 17. The method of claim1, wherein the cell is a native human immature myeloid cell.
 18. Themethod of claim 1, wherein the cell is a myeloid cell and wherein themethod increases the ability of the cell to migrate to endothelial bedsexpressing E-selectin.